Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the peripheral and central nervous systems (CNS) and new ways to effectively control pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send nerve fibers to skin and deep tissues and make connections within dorsal spinal cord, which is the first CNS site of synaptic information processing for pain. The mechanisms of transduction of physical pain stimuli are investigated through models of pathophysiological damage or using reductionist preparations such as primary DRG cultures or heterologous expression systems of ion channels or receptors. Our goals are (a) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (b) to investigate mechanisms underlying human chronic pain disorders, (c) to explore neuronal plasticity and altered gene expression in persistent pain states, and (d) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal through translational research coupled with human clinical trials to develop and introduce new molecular interventions for severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial for in patients with intractable pain from advanced cancer. RTX activates an influx of sodium and calcium ions into nerve endings and once bound to TRPV1, RTX props open the ion channel causing an intracellular calcium cytotoxicity. Depending on the route of administration RTX disables TRPV1 pain-sensing nerve endings or axons (i.e., the nerve fiber) or deletes the neuron entirely. RTX produces very effective pain control in pre-clinical models. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). We have treated 14 patients with pain from advanced cancer. This study is nearly complete unless another, higher, dose tier is investigated. We also published a study of injections around or directly into sensory ganglia, and, based on this approach, we will commence a new protocol for more localized cancer pain problems such as osteosarcoma. Peripheral routes of RTX administration include direct injection into skin, joints, nerve bundles, or topically. Analgesia by these routes is long-lasting, but temporary, since nerve endings and axons regrow. Peripheral administration formed the basis of three reports, one in which we successfully treated experimental burn pain, another in which we successfully used RTX as a preemptive analgesic for surgical incision pain, and a third in which we successfully treated clinical osteoarthritis (OA) pain by intraarticular injection in client owned dogs. The canine results demonstrated both efficacy and a long duration of action (4 to >12 months) and this result is currently being translated to humans. A phase 3 clinical trial will be commencing soon. Early Translational Investigations: In collaboration, we have also examined the pharmacological activity of polyunsaturated ethanolamines and linoleic acid metabolites. In the previous cycle, we evaluated tissue biosynthetic pathways for new endogenous lipids and published our discovery of two previously unknown lipids related to nociception and itch in both animal and human studies. In this cycle we have applied a systems-based approach to characterize oxylipin precursor fatty acids, and the expression of genes coding for proteins involved in biosynthesis, transport, signaling and inactivation of pro- and anti-nociceptive oxylipins in rodent pain circuit tissues. We also measured basal and stimulated levels of predicted oxylipins, throughout the time course of an intraplantar carrageenan injection. These findings advance our understanding of the molecular pathways linking oxylipins and their precursor fatty acids to nociceptive signaling pathways in rats. To extend the systems approach to humans we have established a new human protocol to obtain intraoperative tissue samples from surgical wound margins to perform analyses of lipids and transcriptomic profiles over time. During this cycle we also published a report on a protein therapeutic agent that is a conjugate between Substance P and a bioengineered Pseudomonas exotoxin. This agent is endocytosed by the substance P receptor expressing second order spinal cord dorsal horn neurons and the exotoxin moiety stops protein synthesis thereby killing the neuron and interrupting the pain pathway to the brain. This is a potent analgesic agent. We are currently working with our collaborators to generate high-expressing constructs for SP-PE35 to obtain large amounts of the active pharmaceutical ingredient for further testing in cancer pain and spinal cord injury pain. Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of molecular biology, neuronal function, behavior, and mechanisms of pain transduction. We systematically investigate molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord in order to obtain a comprehensive quantitative foundational molecular understanding of nociceptive processes related to inflammation, surgical incision, and nerve injury. We use a method called RNA-Seq to sequence all of the mRNAs in a given tissue or cell population. Our work now integrates RNA-seq as a component in most of our investigations. We also investigate humans with genetic variations that affect pain sensitivity. At present we are investigating two groups of patients with copy number variants that decrease pain sensitivity. The results are both compelling and informative, and define previously unidentified genetic substrates that can govern pain sensitivity. We also use RNA-seq to define genes involved in inherited peripheral neuropathies and other disorders. These investigations provide new quantitative assessments of neurons of the nociceptive circuit. Through this basic research we aim to obtain a deeper understanding of mechanisms that trigger acute pain and sustain chronic pain and to identify molecular components to control pain.